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Journal of Bacteriology, July 2000, p. 3981-3988, Vol. 182, No. 14
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Rho-Dependent Transcription Termination in the tna Operon of Escherichia coli: Roles of the boxA Sequence and the rut Site

Kouacou Vincent Konandagger and Charles Yanofsky*

Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

Received 23 February 2000/Accepted 28 April 2000

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination. Tryptophan induction prevents Rho-dependent transcription termination in the leader region of the operon. Induction requires translation of a 24-residue leader peptide-coding region, tnaC, containing a single, crucial Trp codon. Studies with a lacZ reporter construct lacking the tnaC-tnaA spacer region suggest that, in the presence of excess tryptophan, the TnaC leader peptide acts in cis on the ribosome translating tnaC to inhibit its release. The stalled ribosome is thought to block Rho's access to the transcript. In this paper we examine the roles of the boxA sequence and the rut site in Rho-dependent termination. Deleting six nucleotides (CGC CCT) of boxA or introducing specific point mutations in boxA results in high-level constitutive expression. Some constitutive changes introduced in boxA do not change the TnaC peptide sequence. We confirm that deletion of the rut site results in constitutive expression. We also demonstrate that, in each constitutive construct, replacement of the tnaC start codon by a UAG stop codon reduces expression significantly, suggesting that constitutive expression requires translation of the tnaC coding sequence. Addition of bicyclomycin, an inhibitor of Rho, to these UAG constructs increases expression, demonstrating that reduced expression is due to Rho action. Combining a boxA point mutation with rut site deletion results in constitutive expression comparable to that of a maximally induced operon. These results support the hypothesis that in the presence of tryptophan the ribosome translating tnaC blocks Rho's access to the boxA and rut sites, thereby preventing transcription termination.

* Corresponding author. Mailing address: Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020. Phone: (650) 725-1835. Fax: (650) 725-8221. E-mail: yanofsky{at}cmgm.stanford.edu.

dagger Present address: Department of Microbiology/Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124.

Journal of Bacteriology, July 2000, p. 3981-3988, Vol. 182, No. 14
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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